Recombinant Thiomicrospira crunogena ATP synthase subunit c (atpE)

What is Thiomicrospira crunogena and why is its atpE protein significant in research?

Thiomicrospira crunogena is a hydrothermal vent gammaproteobacterium that functions as a sulfur chemolithoautotroph, capable of thriving in unstable environments with dramatic changes in habitat chemistry. Originally isolated from the East Pacific Rise, T. crunogena has been detected at deep-sea vents globally in both the Pacific and Atlantic oceans, indicating its ecological importance in these extreme environments .

The atpE protein (ATP synthase subunit c) is a critical component of the F₀F₁-ATP synthase complex responsible for ATP production. In extreme environment organisms like T. crunogena, this protein may contain adaptations that enable energy production under challenging conditions. Studying recombinant atpE provides insights into how these extremophiles maintain energy production despite fluctuating environmental parameters, particularly in low-energy environments like hydrothermal vents.

How is T. crunogena atpE expressed in recombinant systems?

Recombinant expression of T. crunogena atpE protein is typically performed in E. coli expression systems. The full-length protein (amino acids 1-94) is often fused with an N-terminal His tag to facilitate purification . The optimized expression protocol involves:

• Cloning the atpE gene (Tcr_2170) into an appropriate expression vector

• Transformation into competent E. coli cells

• Induction of protein expression under controlled conditions

• Cell lysis and protein extraction

• Purification via affinity chromatography using the His tag

• Final preparation as a lyophilized powder in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0

How does the T. crunogena atpE function compare to other extremophile ATP synthases?

While the search results don't provide direct comparative data, research on ATP synthases from extremophiles suggests that T. crunogena atpE likely contains adaptations that enable function under conditions of variable pH, temperature, and pressure found at hydrothermal vents. The hydrophobic amino acid composition of the protein (evident in its sequence) facilitates its integration into the membrane even under extreme conditions.

T. crunogena has been shown to rapidly adapt to fluctuating environmental conditions, particularly changes in dissolved inorganic carbon (DIC) . Although the specific role of atpE in this adaptation isn't directly addressed in the search results, ATP synthase activity would be essential for maintaining energy production during these rapid physiological shifts.

What is the relationship between T. crunogena's carbon-concentrating mechanism (CCM) and ATP synthase function?

T. crunogena possesses a sophisticated carbon-concentrating mechanism (CCM) that enables it to generate intracellular DIC concentrations 100-fold higher than extracellular concentrations when grown under low-DIC conditions . This mechanism allows the organism to thrive despite environmental CO₂ fluctuations.

The CCM requires energy in the form of ATP to actively transport inorganic carbon against concentration gradients. While the search results don't directly link atpE to CCM function, as a component of ATP synthase, atpE would play a crucial role in generating the ATP necessary to power this energy-intensive adaptation. The transcriptional response studies show that when T. crunogena is exposed to low-DIC conditions, various genes related to energy metabolism are upregulated , suggesting coordinated regulation between carbon acquisition and energy production systems.

How do post-translational modifications affect T. crunogena atpE function in different environmental conditions?

Based on studies of similar systems, ATP synthase subunits can undergo various post-translational modifications that affect their function, including phosphorylation and acetylation. Although the search results don't specifically address post-translational modifications of T. crunogena atpE, research data from similar extremophiles suggests that such modifications could play a role in regulating ATP synthase activity in response to environmental stressors.

The ability of T. crunogena to rapidly respond to changes in its environment (as demonstrated by its transcriptional responses to DIC limitation) indicates sophisticated regulatory mechanisms. While transcriptional regulation has been demonstrated, post-translational regulation of key metabolic enzymes, potentially including ATP synthase components, would provide an additional layer of control for rapidly adapting to environmental fluctuations.

What are optimal protocols for purifying active recombinant T. crunogena atpE protein?

For optimal purification of recombinant T. crunogena atpE protein, the following methodology is recommended:

• Express the His-tagged protein in E. coli using standard induction protocols

• Harvest cells by centrifugation (10,000 × g, 10 min, 4°C)

• Lyse cells using appropriate buffer systems containing protease inhibitors

• Purify using Ni-NTA affinity chromatography

• Further purify by size exclusion chromatography if higher purity is required

• Lyophilize in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

• Store at -20°C/-80°C

For reconstitution, the lyophilized protein should be briefly centrifuged prior to opening, then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% is recommended for long-term storage at -20°C/-80°C .

What experimental approaches can be used to study T. crunogena atpE function in membrane systems?

To study the function of T. crunogena atpE in membrane systems, researchers can employ several experimental approaches:

• Reconstitution into proteoliposomes: Purified atpE can be incorporated into artificial lipid vesicles to study its proton translocation function.

• Patch-clamp electrophysiology: This technique can measure ion conductance through the c-ring formed by multiple atpE subunits.

• FRET-based assays: By labeling the protein with fluorescent probes, conformational changes during function can be monitored.

• Complementation studies: The gene can be expressed in ATP synthase-deficient bacterial strains to assess functional complementation.

• Site-directed mutagenesis: Key residues can be mutated to determine their importance in protein function, particularly those that might be involved in adaptation to extreme conditions.

These approaches can be integrated with the cultivation methods described for T. crunogena, such as the chemostat systems used to study its response to different environmental conditions .

How can researchers effectively measure the impact of environmental variables on atpE expression and function?

Based on methodologies described in the search results for studying T. crunogena's response to environmental changes, researchers can:

• Utilize chemostat cultivation: Grow T. crunogena under controlled conditions where specific variables (pH, temperature, DIC concentration) can be precisely manipulated .

• Implement transcriptional analysis: Use quantitative reverse transcription-PCR (qRT-PCR) to measure changes in atpE gene expression under different conditions, similar to the approaches used to study carboxysome gene expression .

• Perform microarray analysis: Compare genome-wide transcription patterns between different growth conditions to identify co-regulated genes, as was done for DIC and ammonia limitation studies .

• Measure ATP production rates: Quantify the functional output of ATP synthase under different conditions to correlate with atpE expression levels.

• Employ proteomic approaches: Use mass spectrometry to identify post-translational modifications that may occur under different environmental conditions.

The experimental design should include appropriate controls and time-course measurements to capture both rapid responses and long-term adaptations, as T. crunogena has been shown to respond quickly to environmental changes .

What are the common challenges in interpreting functional data for recombinant T. crunogena atpE?

Several challenges exist when interpreting functional data for recombinant T. crunogena atpE:

• Native environment simulation: Hydrothermal vent conditions are difficult to replicate in laboratory settings, potentially affecting protein behavior.

• Protein-lipid interactions: The function of membrane proteins like atpE is heavily influenced by lipid composition, which may differ between T. crunogena and expression hosts like E. coli.

• Complex formation requirements: ATP synthase subunit c functions as part of a larger complex, and studying it in isolation may not reflect its native activity.

• Post-translational modifications: Modifications present in the native protein may be absent in recombinant systems, affecting functional analysis.

• Stability issues: The stability of the recombinant protein may be compromised during purification and storage, as indicated by the recommendation to avoid repeated freeze-thaw cycles .

To address these challenges, researchers should consider complementary approaches, including in vivo studies in the native organism when possible, and careful control experiments in reconstituted systems.

How can researchers distinguish between sequence variations that affect atpE function versus those that are evolutionarily neutral?

To distinguish functional from neutral sequence variations in T. crunogena atpE:

• Comparative genomic analysis: Compare atpE sequences across Thiomicrospira species and related genera to identify conserved versus variable regions. The search results indicate that T. crunogena strains have been detected globally , suggesting potential genetic diversity.

• Structure-function predictions: Use computational modeling to predict how specific amino acid changes might affect protein structure and function.

• Site-directed mutagenesis: Systematically mutate specific residues and assess their impact on protein function in reconstituted systems.

• Evolutionary rate analysis: Calculate the ratio of non-synonymous to synonymous substitutions (dN/dS) to identify regions under selection pressure.

• Heterologous expression: Express variants in ATP synthase-deficient strains to assess functional complementation.

The search results indicate that T. crunogena has adapted to thrive in variable environments , suggesting that certain sequence variations in its proteins, potentially including atpE, contribute to this adaptive capability.

What approaches can integrate atpE functional data with broader studies of T. crunogena metabolism?

Integrating atpE functional data with broader metabolic studies requires multi-level analysis:

• Systems biology approaches: Construct metabolic models that incorporate ATP production rates and requirements for key processes, such as the carbon-concentrating mechanism.

• Multi-omics integration: Combine transcriptomic data (as demonstrated in the DIC limitation studies ) with proteomic and metabolomic analyses to create a comprehensive view of metabolic adaptation.

• Flux analysis: Use metabolic flux analysis to quantify the flow of energy through ATP-dependent pathways under different conditions.

• Comparative physiology: Study how atpE function correlates with whole-organism fitness traits under different environmental conditions.

• Genetic manipulation: Where feasible, create atpE variants or mutants in T. crunogena to directly assess the impact on cellular metabolism.

The search results indicate that T. crunogena responds to nutrient limitation by upregulating novel genes , suggesting that energy metabolism regulation (involving ATP synthase) is integrated with broader adaptive responses.

How does T. crunogena atpE protein contribute to the organism's adaptation to hydrothermal vent environments?

T. crunogena inhabits hydrothermal vent environments characterized by rapid fluctuations in temperature, pH, and available nutrients. As a component of ATP synthase, atpE is crucial for energy production under these variable conditions.

The search results show that T. crunogena has a global distribution at deep-sea vents in both the Pacific and Atlantic oceans , indicating that its energy production systems, including ATP synthase, are robust across diverse hydrothermal environments. The organism's ability to respond rapidly to changes in dissolved inorganic carbon availability would require efficient energy transduction, highlighting the importance of a well-functioning ATP synthase.

While the specific adaptations of atpE aren't detailed in the search results, the protein's amino acid sequence (MEAQFIADIYAATAIGVGVILAAAGLGSAIGWGLICSKTLEGIARQPEMRPALMTNMFIFAGLMESFPFIILAFAMWFLFANPFVGAMQAALGA) contains features typical of membrane proteins that function in extreme environments, including hydrophobic regions that would stabilize it within the membrane.

What novel experimental techniques are emerging for studying extremophile ATP synthases like that of T. crunogena?

Recent advances in experimental techniques for studying extremophile ATP synthases include:

• Cryo-electron microscopy: This technique has revolutionized the structural biology of membrane protein complexes, potentially allowing visualization of T. crunogena ATP synthase under near-native conditions.

• Single-molecule techniques: These approaches can measure the rotary motion and force generation of individual ATP synthase molecules, providing insights into their mechanistic properties.

• Nanodiscs and lipid cubic phase crystallization: These methods provide better membrane mimetics for functional and structural studies of membrane proteins like atpE.

• High-pressure biochemistry: Specialized equipment can now maintain proteins under high-pressure conditions similar to those found at deep-sea vents during functional assays.

• In silico molecular dynamics simulations: Advanced computational methods can model how extremophile proteins like T. crunogena atpE might function under various environmental conditions.

These emerging techniques could help resolve how T. crunogena ATP synthase maintains functionality despite the challenging and variable conditions of hydrothermal vent environments.